Overview

BrdU is a thymidine analogue and when offered to proliferating cells it is incroporated into reduplicating cells. The antibody is specific for DNA in which BrdU has been incorporated. In immunoassays this antibody reacts strongly with free or carrier-protein coupled BrdU but not with other nucleosides. In immuncytochemistry the antibody only recognizes BrdU in denaturated (single stranded) DNA. The BrdU antibody is 100% crossreactive with Iodo-Deoxy-Uridine (IrdU). Therfore, IdU instead of BrdU can be used in studies.

Target

Relevance

The immunocytochemical detection of bromodeoxyuridine (BrdU) incorporated into DNA is a powerful tool to study the cytokinetics of normal and neoplastic cells. In vitro or in vivo labeling of tumor cells with the thymidine analogue BrdU and the subsequent detection of incorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate and comprehensive method to quantitate the degree of DNA-synthesis.
BrdU is incorporated into the newly synthezised DNA of S-phase cells may provide an estimate for the fraction of cells in S-phase. Also dynamic proliferative information such as the S-phase transit rate and the potential doubling time can be obtained, by means of bivariate BrdU/DNA flow cytometric analysis.

Cellular localization

Nuclear

Alternative names

Bromodeoxyuridine antibody

BUdr antibody

Images

ab8152 (1/100) staining BrdU in HeLa cells (green). Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X100/ PBS and counterstained with DAPI in order to highlight the nucleus (red). For further esperimental details please see Abreview.

Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at 25°C; antigen retrieval was by heat mediation in citrate buffer (pH 6). Samples were incubated with primary antibody (1/20 in diluent) for 18 hours at 25°C. A Cy3®-conjugated donkey anti-mouse polyclonal IgG was used as the secondary antibody.

Cells were pulse labeled with 10 mM BrdU for 30 min, rinsed twice in prewarmed PBS, and chased in prewarmed culture medium, supplemented with 5 mM deoxythymidine. Incorporated BrdU was detected after ethanol fixation of the cells, which were than rinsed once in PBS and resuspended in 2 ml of 0.4 mg/ml pepsin in 0.1 N HCl. After 30 min at room temperature cells were pelleted, resuspended in 2 N HCl, and incubated for another 30 min at 37°C. Cells were rinsed in 0.1 M borate buffer, pH 8.5, and PBS/BSA (1 mg/ml BSA in PBS). Appropriately diluted mouse anti-BrdU antibody (clone IIB5) was added to the cell pellet, resuspended in 100 micro liters PBS/BSA. After incubation for 1 h at room temperature, the cells were rinsed twice in PBS/BSA. For visualization, FITC-conjugated Fab2 fragments of rabbit anti-mouse Ig antibody were added in a 1/10 dilution. After incubation for 45 min at room temperature samples were rinsed twice in PBS/BSA and the cells were finally resuspended in 0.5 ml cold PBS supplemented with 100 microgram/ml RNAse and 20 µg/ml propidium iodide. The samples were allowed to stand for 15 min on ice in the dark before flow cytometric analysis. In the negative control the primary antibody was omitted.

I just wanted to follow up on your query with some additional information.

For ab8039, this antibody can be used for detection of BrdU and BrU. We have not tested it regarding other chemicals of this family.

For ab8152, this antibody is specific for DNA in which BrdU has been incorporated. In immunoassays this antibody reacts strongly with free or carrier-protein coupled BrdU but not with other nucleosides. In immuncytochemistry the antibody only recognizes BrdU in denaturated (single stranded) DNA. The BrdU antibody is 100% crossreactive with Iodo-Deoxy-Uridine (IrdU). Therfore, IdU instead of BrdU can be used in studies.

I hope this information helps. Please contact us with any other questions.

For floating sections, please check thefollowing protocol on IHC World website: http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm This protocol is for paraffin-embedded as well as frozen sections.

As for ensuring the paraffin sections stick to your slides: Different sections and tissues stick with differetn strength to the slides. It might be advisable to use charged slides, as well as to bake your sections onto the slides by exposing them to heat (e.g. heating plate or oven) up to 60 degree Celsius.The purpose is to remove miscroscopic amounts of water between the sections and the slide. Subsequently the paraffin would need to removed according to a standard IHC-P protocol. Then you would proceed with the antigen retrieval step.

Also, please check this information on the IHC World website for further advice. http://www.ihcworld.com/_faq/histology-faq/section/s1.htm

The slides need to be immersed, for the retrieval to be effective. Yes, the sections could come off ifthey are kept too long in the retrieval solution. Should they come off, you would need to reduce the time they are exposed to the retrieval buffer.

Are the floating sections paraffin-embedded or frozen? I have found the following protocol on IHC World website which should help you further: http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm

1. Cut and mount sections on slides coated with Vectabond or APES (3-aminopropyltriethoxysilane). 2. Deparaffinize sections and rehydrate to distilled water. 3. Bring 1600ml 0.01M sodium citrate buffer (pH 6.0) to the boil in a Prestige stainless steel pressure cooker, using a hot plate. Cover but do not lock lid. 4. Position slides into metal staining racks and lower into pressure cooker ensuring slides are well immersed in citrate buffer. Lock lid. The small valve will rise. 5. When the pressure indicator valve (the large one) has risen after about 4 minutes, incubate sections for 1 minute. 6. Remove pressure cooker from heat source and run under cold water with lid on. When the small valve sinks open lid and remove slides and place immediately into distilled water. DO NOT OPEN LID UNTIL THE SMALL VALVE SINKS. 7. Wash sections in TBS buffer (pH 7.6) for 1 x 5 minutes. 8. Place sections in 1.5% hydrogen peroxide/methanol for 10 minutes. 9. Wash sections in distilled water for 2 x 5 minutes, then wash sections in TBS buffer for 2 x 5 minutes. 10. Place sections in normal serum for 20 minutes. 11. Cover sections with primary antibody. ( The optimal dilution of the antibody, incubation time and incubation temperature should be determined by the individual laboratory). 12. Wash in TBS buffer for 2 x 5 minutes. 13. Incubate sections in secondary antibody for 30 minutes. 14. Wash in TBS buffer for 2 x 5 minutes. 15. Incubate slides in ABComplex for 30 minutes. 16. Wash in TBS buffer for 2 x 5 minutes. 17. Incubate slides in DAB. 18. Wash in water for 2 x 5 minutes. 19. Counterstain with haematoxylin (if required), dehydrate, coverslip and mount.

To avoid sections becoming detached, sections should be mounted on Vectabond or APES covered slides, then dried at 37oC overnight followed by drying at 56oC for 60 minutes.